I am making tests for diagnostic PCRs. At the moment I am trying to standardize a pcr reaction for a circa 180 bp product. I did not get positive bands so far. Interestingly, I get what appears to be a primer smear in both negative and positive controls. But no smear at all for DNA samples I do not know abou pathogen absence/presence. Any idea why this is happenning?
I think that many factors contribute. primer dimer is worse at high primer concentartions and high magnesium. In the no dna control the MG and primer concentrations are as high as it is possible to be and the enzyme has nothing else to do except amplify the PD so we get very high amounts of PD.
In the nehative samples there is dna which allows primer binding randomly so lessening the free primer concentrations and the dna removes some MG and the enzyme does do some work amplifying mismatched primers giving random lengths of amplimer off each primer annealing in the wrong places but these amplifications are linearly produced not logarithmically produced so their products are too faint to be seen on a gel but primer is removed so lessening the amount of PD
There are tools to help screen (emphasize screen) for primer interactions. They may help predict primer dimer formation (remember they are not 100% sensitive & specific) which you can combine with the wet-lab evidance you have to make judgement:
https://www.thermofisher.com/tr/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/multiple-primer-analyzer.html
Can you post a picture of your gel and what is your positive control (how does that template differ from the test samples)? If you can get/make a plasmid with your target gene in it that would make a useful positive control. Keep in mind that PCR on pure plasmid will be far more efficient than PCR from other template types (gDNA, colony PCR and so forth), but it will at least let you validate your primers.
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